2. Materials and methods
2.1. Preparation of buffers The following four buffers for cryopreservation of buffalo spermatozoa were used.
Ž .
1. CITRATE. tri-Sodium citrate dihydrate Merck, Darmstadt, Germany 6.37 g in 200 ml distilled H O, pH 6.8, 301 mOsm kg
y1
.
2
Ž .
Ž 2. TCA. Citric acid Fluka, Switzerland 12.50 g in 200 ml distilled H O 325 mOsm
2 y1
. Ž
. Ž
. Ž kg
was titrated to Tris- hydroxymethyl -aminomethane Fluka, Switzerland Tris;
y1
. 7.87 g in 200 ml distilled H O, 325 mOsm kg
to pH 7.0. Osmotic pressure was
2
320 mOsm kg
y1
. Ž
w x
. Ž 3. TEST. Tes N-tris Hydroxymethyl methyl-2-aminoethane-sulfonic acid
Sigma, St. .
Ž
y1
. Louis, MO 14.89 g in 200 ml distilled H O 325 mOsm kg
and was titrated to
2
Ž .
Tris 7.87 g in 200 ml distilled H O to pH 7.0. Osmotic pressure was 303 mOsm
2
kg
y1
. Ž
w x
w x. Ž
. 4. HEPEST. Hepes
N- 2-Hydroxyethyl piperazine-N- 2-ethanesulfonic acid Sigma
Ž
y1
. Ž
15.49 g in 200 ml distilled H O 325 mOsm kg was titrated to Tris 7.87 g in 200
2
.
y1
ml distilled H O to pH 7.0. Osmotic pressure was 301 mOsm kg .
2
Ž .
Ž .
Ž .
Ž Egg yolk 20; vrv , fructose 0.2; wrv , glycerol 6; vrv , penicillin 1000
y1
. Ž
y1
. I.U. ml
and streptomycin 100 mg ml were added to each of the formulated
buffer. The extenders were centrifuged at 12,000 = g for 15 min, and the supernatant was frozen and stored at y208C. The extenders were thawed at 378C for experimental
use.
2.2. Semen collection and initial eÕaluation Four mature Nili–Ravi buffalo bulls, maintained at Livestock Research Station,
National Agricultural Research Centre, Islamabad, Pakistan, were used in the study. Ž
. Two consecutive ejaculates were collected with the help of artificial vagina 428C at
Ž .
weekly intervals for 5 weeks replicates during the months of May and June. The semen was transferred to laboratory within a minute. Visual motility was assessed microscopi-
Ž .
Ž .
cally =400 with closed circuit television Graham et al., 1970 . Sperm concentration Ž
. was assessed by digital-photometer Dr. Lange LP 300 SDM; Germany at 560 nm. The
neat semen samples with more than 60 motile spermatozoa were used. The qualifying ejaculates were pooled in order to have sufficient semen for a replicate and to eliminate
the bull effect. The semen was given a holding time of 15 min at 378C in the water bath before dilution.
2.3. Semen processing The schematic diagram of cryopreservation of buffalo spermatozoa are presented in
Fig. 1. Four aliquots of semen were diluted at 378C with each extender in order to provide approximately dilution rate of 50 = 10
6
spermatozoa ml
y1
. Extended semen
Fig. 1. The schematic diagram of cryopreservation of buffalo semen.
Ž .
was cooled slowly approximately in 2 h to 48C, and equilibrated for 4 h. Semen was packed into 0.5 ml polyvinyl French straws and frozen in a programmable cell freezer
Ž .
KRYO 10 Series III, Planer, Sunbury-on-Thames, Middlesex, UK from 48C to y158C at the rate of 38C min
y1
and from y158C to y808C at the rate of 108C min
y1
. Ž
. Semen-filled straws were plunged into liquid nitrogen y1968C for storage. After 24 h
storage in liquid nitrogen, semen straws were thawed at 378C for at least 15 s for post-thaw semen quality assessment.
2.4. Semen assays 2.4.1. Visual assessment
Ž .
Ž .
Visual motility VMOT, was assessed as described earlier Section 2.2 . 2.4.2. Sperm motion characteristics
2.4.2.1. The settings of CASA. Sperm motion characteristics were determined using the Ž
. Cell Motion Analyzer SM-CMA version 4.4; Mika Medical, Germany as previously
Ž .
reported in bulls Engelmann et al., 1992 . The system included an IBM-compatible PC computer with a monitor, a Panasonic KX-P1121 multimode printer, and an Olympus
BH-2 microscope equipped with a thermostage, video camera and Panasonic BT-H1450Y digital image video monitor. The video images were fed into IBM-compatible PC
through video camera and monitor, which analysed the data according to the pro- grammed algorithms in SM-CMA software. The settings of the computer were adjusted
to acquire digitized video scan data at the rate of 32 frames s
y1
. Lower area detection limit was 35 pixels and upper area limit was 300 pixels. Upper velocity limit for
immotile spermatozoa was 20 mm s
y1
. Lower velocity limit for the motile spermatozoa was 30 mm s
y1
. The spermatozoa moving with velocity between 20 and 30 mm s
y1
were considered as locally motile and were not included in the motile category. The spermatozoa moving with a radius of 10 mm were designated as circular.
2.4.2.2. Validation of CASA. The system was validated before use for percentage of motile spermatozoa to evaluate the accuracy for the absolute standard in buffalo. Semen
Ž .
of four buffalo bulls were pooled n s 5 and diluted at 378C with sodium citrate in order to provide approximately 50 = 10
6
spermatozoa ml
y1
. Extended semen was cooled slowly in 2 h to 48C. For the assessment of sperm motion characteristics, cooled
Ž .
semen was divided into two parts. One part of the semen remained as such live and, Ž
. the other part was repeatedly plunged into liquid nitrogen and thawed killed . Five
mixtures of live and killed spermatozoa were prepared with 100:0, 75:25, 50:50, 25:75 Ž
. and 0:100 ratio live:killed by volume. Correlation between the percentage of motile
Ž Ž
.. and the addition of killed spermatozoa was significant r s 0.98; P - 0.05; Fig. 2 .
The straight-line velocities of these mixtures were 52.6 4.0, 55.6 2.8, 54.0 2.4, Ž
. 52.0 3.1, and 0.0 0.0 mean SEM , respectively.
2.4.2.3. Variables determined using CASA. Frozen semen was thawed at 378C for 15 s and maintained at the same temperature for 5 min before evaluation of motion
characteristics. After thoroughly mixing, 5 ml semen sample was placed on a prewarmed Ž
. Ž
. 378C Makler Chamber depth 10 mm; Sefi-Medical Industries, Haifa, Israel . The
Ž .
chamber was then placed on the prewarmed stage 378C of microscope connected with Ž
. CASA. Sperm motion characteristics included computer-assisted motility CMOT, ,
Ž .
Ž .
Ž linear motility LMOT, , circular motility CIRMOT, , straight-line velocity VSL,
mm s
y1
; the straight-line distance from beginning to end of track divided by time .
Ž
y1
taken , average path velocity VAP, mm s ; the spatially averaged path that eliminated
. Ž
y1
the wobble of the sperm head , curvilinear velocity VCL, mm s ; a measurement of
Fig. 2. Percentage decline in sperm motility after addition of killed spermatozoa in buffalo bulls.
. the total distance traveled by a sperm during the acquisition divided by the time taken ,
Ž lateral head displacement LHD, mm; deviation of the sperm head about the axis of
. Ž
. Ž
average path , linearity LIN s VSLrVCL = 100 , and straightness STR s VSLrVAP .
Ž =
100 . Each observation was based upon the average of three different fields Anzar et .
al., 1991 . 2.4.3. Hypo-osmotic swelling HOS .
Ž .
Plasma membrane integrity PMI of spermatozoa was assessed using the HOS-assay as Ž
. described earlier Jeyendran et al., 1984 . HOS solution was prepared by dissolving
Ž 0.735 g sodium citrate and 1.351 g fructose in 100 ml distilled H O osmotic pressure
2 y1
. Ž
. ;
190 mOsm kg , according to World Health Organization 1992 manual. The HOS
assay was performed by mixing 50 ml of the semen samples to 500 ml of the prewarmed Ž
. 378C HOS solution and incubated at 378C for 30–45 min. After incubation, a drop of
Ž .
semen sample was examined under phase-contrast microscope =400 . Two hundred spermatozoa were counted for their swelling, characterized by coiled tail, indicating
intact plasma membrane.
2.4.4. Normal acrosomes. Ž
. Ž
. Semen sample 500 ml was fixed by 50 ml of 1 formal citrate Hancock, 1957 which
Ž .
was prepared by adding 1 ml of 37 commercial formaldehyde Merck in 99 ml of Ž
. 2.9 wrv sodium citrate. Acrosomal integrity characterized by normal apical ridge
Ž .
Ž .
NAR was assessed under oil immersion =1000 using phase-contrast microscope Ž
. Leica, Leitz Wetzlar, Germany . Two hundred spermatozoa were counted.
2.5. Statistical analysis Results in text are presented as mean SEM. Data for each variable were analysed
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